Assembly for gel casting: Take clean plates and the spacers. Snuggly tighten them to make casting module. Fit it on the stand without the rubber spacer on bottom. Add some water to make sure water is not leaking out at the bottom. Keep the comb handy. Drain off the water by inverting the gel cast. Few droplets sticking to glass plates don't interfere with gel.
Gel solution preparation and casting:
Water distilled 50 ml
30:2 acralamide: bis 8 ml
10X TBE 1.5 ml
10% APS 600 ul (0.0600 gm in 600 ul)
Temed 60 ul
Add temed at last. Mix well. Now gel is ready to pour.
Take 25ml pipette and try to pour the gel on the side of cast near spacers. So it trickles on the side and fills from bottom without any air bubbles trapped inside. Fill it up to the top. Take comb and insert from one side keeping angle so that each tooth of comb can be inserted one by one in Gel without trapping any air bubble. The gel should polymerize in half and hour and ready to use. Make sure its not leaking from anywhere when you leave it to polymerize.Oligoneucleotide Labeling
We want our final labeled product to be 4.8 pm/ul so dilute initial single stranded oligoneculeotide at 480 pm/ul. Now we need to anneal them.
- 100 ul of annealing buffer ( 10mm Tris-HCl, Ph 7.5, 20mm NaCl)
- 1 ul or each primer (480 pm of each of them)
- Heat it for 95C for 5 min on heat block. Leave the block at RT afterwards.
- Store at ¨C20* freezer once cooled down.
- Labeling with 32P
Incubate at 37* for 30 minutes
2 ul of oligo
1 ul of PNK buffer
4 ul of water
1 ul T4- Kinase
2 ul of 32P ATP
Add 40 ul of distilled water
Now we want to remove unlabelled oligos. We use Quick Spin Column (TE) for Radiolabelled DNA (Roche) for it.
Keep column at room temp for half an hour. Remove the cap first and then bottom
Seal to avoid generating suction. Let the supernatant drain off with gravity.
Add 50 ul of your reaction.
Spin them around 1000xg (2200 RPM for the machine in tissue culture room) for 4 minutes with tubes provided with column to collect labeled product.
Make final volume of 100 ul by adding water.
Now we want to initiate reaction between labeled oligos and nuclear extract of the cell line of your interest.
These ingredients go in each tube. Incubate on them ice for 10 min.
Add cold competitor in designated tubes ( 2 ul or more according to your need)
Incubate on ice for 10 min
Add hot probe 1ul in designated tubes and incubate for RT for 20 min or on ice for 45 min.
Add 2 ul of loading dye to stop reaction.
You are ready to load them on Gel.
Before we can run the samples we need to pre-run it for 30 min at 80 volts.
Prepare the buffer 0.5X TBE for running.
Load the samples one by one. Making sure not to spill in upper chamber (buffer will become radioactive otherwise)
Run the gel @ 120 V for 2-3 hours. When dye front is reaching lower fifth of gel. Stop the reaction. (Again if you let it long, lower chamber buffer will be radioactive).
Gel drying and imaging
Decant the buffer after making sure it is not radioactive in to the sink. Otherwise collect it in radioactive liquid waste container.
Get the gel cast out. Separate the glass plate carefully so gel sticks to either of the plate. Cut whatman size 3 papers to size of gel. Put couple on it. Gel should adhere to it and will be easy to lift off.
Now we want it to be dried. Cover it with cellophane. Dry it for ~ 40 min at 80* C. Keep the suction on and turn of the temp for 15 min. Disconnect the suction and turn off the pump. Now gel is dried and ready for imaging.
We use Biomax MR Kodak films for it. It is single sided so orientation is important. Put the gel in cassette and take individually rapped film to dark room. Open the film in dark room. Put it on the gel with notch on film on upper right hand or lower left hand. Close the cassette. You can leave it at RT for 7-8 hrs or in ¨C80 for 4-5 hrs. Take it back to dark room. Open the cassette and develop the film.
Preparation of nuclear extract
- Wash cells with PBS two times. (Take about 107 cells)
- For adherent cells wash cells and then after scrapping them spin them down. Store on dry ice or ¨C80* till all cells are ready for further steps.
- Add 200 ul of Buffer A / 107 cells. Pipette up and down till no clumps remain. To prepare buffer A take 2.4875 ml of stock solution of buffer A and add 12.5 ul of DTT before use.
- Incubate on ice for 10 min. Mix occasionally by flicking with finger.
- Spin full speed 10 sec at RT.
- Remove suspension. Remember this protocol leaves cytoskeletal proteins with nuclear extract. Use another protocol to get those proteins separately.
- Add double the amount of pallet buffer C. To prepare buffer C, take 2.1375 ml of stock solution and add 12.5 ul of DTT and 350 ul of cocktail protease inhibitors. Flick the tube, don¡¯t pipette up and down.
- Incubate 30 min on ice.
- Spin 30 min full speed at 4* C. (cold room)
- Transfer the supernatant. It should not be sticky. Measure the protein concentration.